Commonly, drug absorption and transport across the blood brain barrier (BBB) or gastrointestinal tract (GIT) is studied via in vitro permeability assays. These assays are typically performed in an apparatus featuring solution filled donor and acceptor compartments separated by a porous support such as a microporous material or structure. A molecular entity such as a drug of interest permeates from the donor compartment into the acceptor compartment through the porous support of the apparatus. For example, to model GIT drug absorption and transport, monolayers of living cells are grown on the porous support to form a lipid membrane permeation barrier. These monolayers often can include Caco-2 type cells. Alternatively, a lipid membrane permeation barrier can be formed by depositing biomimetic materials on or into the porous support for an assay that is often referred to as a parallel artificial membrane permeability assay (PAMPA).
The donor and acceptor compartments of the apparatus are usually incorporated into microtitre plates of various formats to conduct numerous assays simultaneously. The shortcoming with the compartments is that their small volume causes thick aqueous boundary layers of stagnant solution adjacent to the porous support comprising, for example, biological or biomimetic materials. A thick boundary layer along the upper or lower surface of the support can introduce significant errors to permeation measurements. For example, a molecular entity will be physically impeded from passing through the porous support, which comprises a biological or biomimetic material to form a permeation barrier, from the donor to acceptor compartment.
With a conventional PAMPA, the total thickness of the aqueous boundary layers adjacent to the upper and lower surface of the porous support is from about 1,500 to 4,000 microns (μm). Measurements of, for example, a drug through a permeation barrier in such an assay can be appreciably biased by resistance due to the boundary layers. Indeed, drug studies are replete with lipophilic compounds that have reported permeability values that merely represent that of boundary layers characteristic to a given acceptor or donor compartment.
In vivo GIT boundary layers are usually considered to be from about 30 to 100 μm thick. Moreover, BBB boundary layers are presumed to be less than 1 μm. With a standard Caco-2 type cell assay or PAMPA, the total thickness of the aqueous boundary layers adjacent to the porous support is commonly more than 1,500 μm thick such that the layers tend to become a limiting factor when measuring the permeability of lipophilic compounds. For such assays, it is well established that solution agitation can diminish aqueous boundary layer thickness. A common approach to achieve agitation of solution in the donor and acceptor compartments is to place the compartments on a vibrational body such as an orbital or linear plate shaker. Other approaches for reducing aqueous boundary layer thicknesses include using a chemical sink in the acceptor compartment or to induce a pH gradient across the porous support comprising, for example, biological or biomimetic materials.
These approaches are still unable to reduce boundary layer thicknesses below 300 to 500 μm, which is necessary to closely model biological conditions, for example, for drug absorption and transport. Moreover, such approaches become even less attractive for donor and acceptor compartments incorporated into microtitre plates. For example, a standard 96 compartment microtitre plate exhibits a high degree of anisotropy across the plate with compartments along its edges being more effectively agitated than those near the center. This sort of anisotropy is even more pronounced in higher density plates such as those with 384 or 1536 compartments.
In view of the interest in using high density microtitre plates to conduct numerous permeation assays simultaneously, minimizing aqueous boundary layer thicknesses is increasingly difficult as smaller donor and acceptor compartments require more vigorous agitation to only marginally reduce boundary layer thickness. Such a shortcoming as well as those mentioned above demonstrate the need to have a convenient means by which to reduce aqueous boundary layer thicknesses in permeation assays. The means should also be adaptable to easily modify a standard Caco-2 type cell assay or PAMPA for in vitro studies of drug absorption and transport.